EP3442993A1 - Methods and kits for the rapid detection of the escherichia coli o25b-st131 clone - Google Patents
Methods and kits for the rapid detection of the escherichia coli o25b-st131 cloneInfo
- Publication number
- EP3442993A1 EP3442993A1 EP17718047.8A EP17718047A EP3442993A1 EP 3442993 A1 EP3442993 A1 EP 3442993A1 EP 17718047 A EP17718047 A EP 17718047A EP 3442993 A1 EP3442993 A1 EP 3442993A1
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- European Patent Office
- Prior art keywords
- polypeptide
- bacteriophage
- strains
- clone
- coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10232—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
Definitions
- the present invention relates to methods and kits for the rapid detection of the Escherichia coli 025b-ST131 clone.
- Escherichia coli 025b-ST131 clone is a worldwide pandemic clone, causing predominantly community-onset antimicrobial-resistant infection. For example, a high prevalence of the clone ( ⁇ 30%-60%) has been identified amongst fluoroquinolone-resistant E. coli. In addition, it potentially harbours a variety of ⁇ -lactamase genes; most often, these include CTX-M family ⁇ -lactamases, and, less frequently, TEM, SHV and CMY ⁇ - lactamases. A broad distribution has been demonstrated amongst antimicrobial-resistant E. coli from human infection in Europe (particularly the UK), North America, Canada, Japan and Korea. The clinical spectrum of disease described is similar to that for other E.
- E coli 025b-ST131 clone has also been reported as a prominent cause of E. coli neonatal sepsis.
- the clone thus constitutes a major public health concern and there is an unmet need for the development of new method for detecting it all the more than phenotypic detection of the ST131 clone is not possible and DNA-based techniques, including MLST and PCR, remains time consuming.
- the present invention relates to methods and kits for the rapid detection of the Escherichia coli 025b-ST131 clone.
- the present invention is defined by the claims.
- the inventors have isolated a podoviridae bacteriophage (LM33 PI) infecting the E. coli strain LM33 isolated from ventilator associated pneumonia and which belongs to clone STB 1 -025b. Out of 70 025b strains tested, LM33 P 1 was able to infect 73% of them. By testing different strains of E coli belonging to 129 others various distinct serotypes (including twelve 025a) the inventors found that bacteriophage LM33 P 1 is able to infect exclusively 025b strains (none of non-025b strains could be infected by LM33 P 1 ).
- LM33 PI podoviridae bacteriophage
- the inventors have determined that the specificity displayed by bacteriophage LM33 P 1 to infect only 025b serotype strains is based on a very specific polypeptide (Gp l 7) used by I. 33 P 1 to attach the bacterial cell via LPS molecule.
- a first object of the present invention relates to a polypeptide comprising an amino acid sequence having at least 80% of identity with the amino acid sequence set forth in SEQ ID N0: 1.
- a first amino acid sequence having at least 80% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100%) of identity with the second amino acid sequence.
- Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv.
- ALIGN Myers and Miller, CABIOS 4: 11-17, 1989
- LFASTA Nearson and Lipman, 1988
- ALIGN compares entire sequences against one another
- LFASTA compares regions of local similarity.
- the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
- the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
- the BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. EnzymoL, 266: 131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.
- the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: l .
- polypeptide of the present invention is a functional conservative variant of the polypeptide consisting of SEQ IID NO: l .
- function- conservative variant are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
- a “function-conservative variant” also includes a polypeptide which has the same or substantially similar properties or functions as the native or parent protein to which it is compared (i.e. binding to the LPS molecules of the E coli 025b-ST131 clone.). Functional properties of the polypeptide of the present invention could typically be assessed in any functional assay as described in EXAMPLE.
- the polypeptide of the present invention is fused to at least one heterologous polypeptide (i.e. a polypeptide which is not derived from SEQ ID NO: l) to create a fusion protein.
- fusion protein refers to the polypeptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.
- the fusion protein comprises the polypeptide according to the invention that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.
- the term "directly" means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the polypeptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.
- the last amino acid of the C-terminal end of said polypeptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said polypeptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.
- spacer refers to a sequence of at least one amino acid that links the polypeptide of the present invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances.
- the linker is typically a spacer peptide and will, according to the invention, be selected so as to allow binding of the polypeptide to the heterologous polypeptide.
- Suitable spacers will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine experimentation. Suitable spacers are described herein and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 2 or more amino acids.
- the spacer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.
- the upper limit is not critical but is chosen for reasons of convenience regarding e.g. production of such fusion proteins.
- One useful group of spacer sequences are spacers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678.
- Other examples are poly- alanine spacer sequences such as Ala-Ala-Ala.
- spacer sequences are Gly/Ser spacers of different length including (gly4ser)3 , (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.
- the polypeptide of the present invention is fused to an immunoglobulin domain.
- the fusion protein of the present invention may comprise a polypeptide of the present invention that is fused to an Fc portion (such as a human Fc). Said Fc portion may be useful for increasing the production of the polypeptide of the present invention or for mobilizing the polypeptide of the present invention to a solid support.
- the polypeptide of the present invention is fused to one or more (typically human) CHI, and/or CH2 and/or CH3 domains, optionally via a spacer sequence.
- polypeptide of the present invention fused to a suitable CHI domain could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')2 fragments, but in which one or (in case of an F(ab')2 fragment) one or both of the conventional VH domains have been replaced by a polypeptide of the present invention.
- one or more single domain antibodies of the invention may be fused to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions and/or may confer the ability to bind to one or more Fc receptors.
- the one or more further amino acid sequences may comprise one or more CH2 and/or CH3 domains of an antibody, such as from a heavy chain antibody and more typically from a conventional human chain antibody; and/or may form and Fc region, for example from IgG (e.g. from IgGl , IgG2, IgG3 or IgG4), from IgE or from another human Ig such as IgA, IgD or IgM.
- the heterologous polypeptide is a fluorescent polypeptide.
- Suitable fluorescent polypeptides include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a "humanized” version of a GFP, e.g., wherein codons of the naturally-occurring nucleotide sequence are changed to more closely match human codon bias; a GFP derived from Aequoria victoria or a derivative thereof, e.g., a "humanized” derivative such as Enhanced GFP, which are available commercially, e.g., from Clontech, Inc.; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al.
- GFP green fluorescent protein
- the heterologous polypeptide is an enzyme.
- said enzyme may be selected from the group consisting of ⁇ -galactosidase, alkaline phosphatase, luciferase, and horse radish peroxidase).
- the heterologous polypeptide is an enzyme that yields a detectable product
- the product can be detected using an appropriate means, e.g., ⁇ -galactosidase can, depending on the substrate, yield colored product, which is detected spectrophotometrically, or a fluorescent product; luciferase can yield a luminescent product detectable with a luminometer; etc.
- the heterologous polypeptide is a polypeptide that facilitates purification or isolation of the fusion protein, e.g., metal ion binding polypeptides such as 6H is tags (e.g., acetylated Tat/6His), or glutathione-S-transferase.
- metal ion binding polypeptides such as 6H is tags (e.g., acetylated Tat/6His), or glutathione-S-transferase.
- polypeptide of the present invention (fused or not to the heterologous polypeptide) is produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
- any technique known in the art such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
- one skilled in the art can readily produce said polypeptide (fused or not to the heterologous polypeptide), by standard techniques for production of polypeptides. For instance, they can be synthesized using well- known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions.
- the polypeptide of the present invention can be synthesized by recombinant DNA techniques well-known in the art.
- the polypeptide of the present invention can be obtained as DNA expression products after incorporation of DNA sequences encoding the polypeptide (fused or not to the heterologous polypeptide) into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
- a variety of expression vector/host systems may be utilized to contain and express the polypeptide of the present invention (fused or not to the heterologous polypeptide).
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al, 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al, 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al, 2000); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al, 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et
- the expression vectors include DNA encoding the given protein being operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes.
- suitable transcriptional or translational regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation.
- a suitable expression vector for expression of polypeptide of the present invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan.
- the nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the protein of interest (e.g., a polypeptide).
- a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulphate precipitation, electrophoresis, gel filtration, affinity chromatography, etc.
- control sequences' refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
- nucleic acid sequence is a "promoter” sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
- Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
- a further object of the present invention relates to a nucleic acid molecule which encodes for a polypeptide of the present invention (fused or not to the heterologous polypeptide).
- nucleic acid molecule has its general meaning in the art and refers to a DNA or RNA molecule.
- the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5 -carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine,
- 2- thiocytosine 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
- the nucleic acid molecule of the present invention is thus included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
- a further object of the invention relates to a vector comprising a nucleic acid encoding for a polypeptide of the present invention (fused or not to the heterologous polypeptide).
- a further object of the present invention relates to a host cell transformed with the nucleic acid molecule of the present invention.
- transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, i.e. the polypeptide encoded by the introduced gene or sequence.
- a host cell that receives and expresses introduced DNA or RNA has been "transformed”. For instance, as disclosed above, for expressing and producing the polypeptide of the present invention, prokaryotic cells and, in particular E. coli cells, will be chosen.
- the host cell may be suitable for producing the polypeptide of the present invention (fused or not to the heterologous polypeptide) as described above.
- the polypeptide of the present invention (fused or not to the heterologous polypeptide) is conjugated with a detectable label.
- detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bio luminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
- the detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are 3 H, 125 I, 131 I, 35 S and 14 C.
- the polypeptide of the present invention (fused or not to the heterologous polypeptide) can also be labeled with a fluorescent compound.
- the presence of a fluorescently-labeled polypeptide of the present invention is determined by exposing the immuno conjugate to light of the proper wavelength and detecting the resultant fluorescence.
- Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine and Alexa Fluor dyes.
- the polypeptide of the present invention can be detectably labeled by coupling said polypeptide to a chemiluminescent compound.
- the presence of the chemiluminescent-tagged immuno conjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
- chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
- a bio luminescent compound can be used to label the polypeptide of the present invention.
- Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction.
- the presence of a bioluminescent protein is determined by detecting the presence of luminescence.
- Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
- the presence of the fusion protein can be detected with any means well known in the art such as a microscope or microscope or automated analysis system.
- the fusion protein is incubated in the presence of the appropriate substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
- polyspecific immunoconjugates examples include ⁇ -galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
- enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
- suitable labels which can be employed in accordance with the present invention.
- the binding of marker moieties to anti- the polypeptide of the present invention is accomplished using standard techniques known to the art. Typical methodology in this regard is described by Kennedy et al., Clin. Chim. Acta 70: 1, 1976; Schurs et al, Clin. Chim. Acta 81 : 1, 1977; Shih et al, Int'U. Cancer 46: 1101, 1990; Stein et al, Cancer Res.
- the presence of the polypeptide (fused or not to the heterologous polypeptide) is detected with a secondary antibody that is specific for the single antibody of the present invention (fused or not to the heterologous polypeptide).
- a secondary antibody that is specific for the single antibody of the present invention (fused or not to the heterologous polypeptide).
- said secondary is labeled by same methods as described above.
- the polypeptide of the present invention is fused to a tag (e.g. histidine tag) the secondary antibody is specific for said tag.
- the polypeptide of the present invention is biotinylated.
- biotinylation refers to the covalent binding of biotin to a polypeptide.
- the biotinylation of the polypeptide of the present invention is carried out using reagents capable of conjugating biotin to the side chain of said polypeptide, wherein said conjugation fundamentally takes place in the primary amino groups and in the thiol groups appearing in the side chains of the polypeptide.
- Suitable reagents for the biotinylation of amino groups include molecules containing biotin and a group capable of reacting with amino groups such as succinimide esters, pentafluorophenyl ester or alkyl halides, the biotin group and the reactive group being separated by a spacer of any length (for example, of 8-40 A in length).
- biotinylation agents include NHS-biotin agents (containing an ester bond of five carbon atoms between the biotin and the NHS group), sulfo-NHS-biotin, NHS- LC-biotin, sulfo-NHS-LC-Biotin, NHS-LC-LC-biotin, sulfo-NHS-LC-LC-biotin, sulfo-NHS- SS-biotin, NHS-PE04-biotin, PFP-biotin, TFP-PEO-biotin and the like, wherein “NHS” indicates a N-hydroxysuccinimide group, "LC” refers to an amide type bond of 6 carbon atoms located between the NHS group and the biotin, "PEO” refers to a ethylene oxide group, wherein the subscript indicates the number of PEO units, "PFP” refers to a pentafluorophenyl group, "TFP” refers to a tetra
- biotinylation reactive agents with thiol groups include molecules comprising biotin and a group of the maleimido or alkyl halide type, separated by a spacer of any length.
- biotinylation reagents include maleimide-PEG-biotin, biotin-BMCC (containing an N- terminal maleimido group and a cyclohexyl group, 2 amide bonds and 9 linking carbon atoms), PEO-iodoacetyl biotin, iodoacetyl-LC-biotin, biotin-HPDP (containing a pyridyl disulfide group) and the like.
- the polypeptide of the present invention is conjugated to a latex particle, a metal colloid particle, or a carbon nanotube.
- One further object of the present invention relates to a method for detecting the presence of the E coli 025b-ST131 clone in a sample comprising i) contacting the sample with the polypeptide of the present invention which is capable of forming complexes with the lipopolysaccharide (LPS) molecules of the E coli 025b-ST131 clone and ii) detecting the presence of the one or more said complexes wherein the presence of at least one complex indicates the presence of the clone in the sample.
- LPS lipopolysaccharide
- sample encompasses a variety of sample types obtained from a subject and can be used in an assay and susceptible to contain the E coli 025b-ST131 clone.
- Biological samples include but are not limited to blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
- the sample is a liquid sample.
- the liquid sample is urine, blood, serum, blood products, plasma, saliva, body fluid, water, culture medium, diluted culture medium, petroleum product, fuel, liquid undergoing fermentation, or a beverage.
- the sample is a solid sample.
- the solid sample is human or animal tissue, stool, sputum, expectorate, an agricultural product, food, solids collected by centrifugation or filtration, soil, or sediment.
- the sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise manipulated prior to assay.
- the detection of the complexes may be performed by any well known method in the art and typically include immunoassays.
- Immunoassay formats that can be used typically include an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IF A), a radioimmunoassay (RIA), a chemiluminescence immunoassay (CLIA), a lateral flow chromatographic test, a Western blot, an immunoprecipitation assay, flow cytometry, or fluorescence microscopy. Lateral flow immunochromatographic tests are particularly suitable for the rapid detection of the clone. Those skilled in the art also know that such assays may be useful for any of a large number of clinical microbiology problems, and other types of clinical samples. For performing said immunoassays the polypeptide is thus typically immobilised on a solid support.
- the polypeptide of the present invention is immobilized on a solid support.
- the solid support is a particle, a bead, a plastic or glass surface, a porous membrane, an array, or a chip.
- the solid support forms part of an assay device. Solid phase assays, in general, are easier to perform than heterogeneous assay methods which require a separation step, such as precipitation, centrifugation, filtration, chromatography, or magnetism, because separation of reagents is faster and simpler.
- the present invention provides devices that are useful to detect and/or visualize the presence of the E coli 025b-ST131 clone in a sample. These devices may comprise a surface and the polypeptide of the present invention.
- Solid-phase assay devices include microtiter plates, flow-through assay devices (e.g., lateral flow immunoassay devices), dipsticks, and immunocapillary or immunochromatographic immunoassay devices.
- a particularly useful assay format is a lateral flow immunoassay format.
- the device includes a solid support that contains a sample application zone and a capture zone.
- the lateral flow immunoassay (LFA) is a particular embodiment that allows the user to perform a complete immunoassay within 10 minutes or less.
- lateral flow format including: a variety of porous materials including nitrocellulose, polyvinylidene difluoride, paper, and fiber glass; a variety of test strip housings; colored and fluorescent particles for signal detection including colloidal metals, sols, and polymer latexes; a variety of labels, binding chemistries, and other variations.
- One format of LFA uses a direct binding "sandwich" assay, wherein the analyte is bound by two specific binding molecules which can thus include one polypeptide of the present invention.
- the polypeptide of the present invention is fused to Fc domain (as described above) to form a antibody-like molecule than can be attached to a solid support by any method well known in the art.
- Fc domain as described above
- Examples of LFA format are described in U.S. Pat. No. 4,861,711; H. Friesen et al. (1989), which discloses a solid-phase diagnostic device for the determination of biological substances; U.S. Pat. No. 4,740,468; L. Weng et al. (1988) which discloses a solid phase specific binding method and device for detecting an analyte; U.S. Pat. No. 4,168,146; A. Grubb et al.
- target analyte i.e. LPS molecules
- capture reagent such as labeled (e.g., gold-conjugated or colored latex particle-conjugated protein).
- detector reagent such as labeled (e.g., gold-conjugated or colored latex particle-conjugated protein).
- the detector reagent may be placed on the membrane in a manner that permits the detector to mix with the sample and thereby label the analyte.
- the visual detection of detector reagent provides an indication of the presence of target analyte in the sample. Representative flow-through assay devices are described in U.S. Pat. Nos.
- Migration assay devices usually incorporate within them reagents that have been attached to colored labels, thereby permitting visible detection of the assay results without addition of further substances. See, for example, U.S. Pat. No. 4,770,853; PCT Publication No. WO 88/08534 and European Patent No. EP-A 0 299 428.
- U.S. Pat. No. 5,229,073 describes a semiquantitative competitive immunoassay lateral flow method for measuring plasma lipoprotein levels. This method utilizes a plurality of capture zones or lines containing immobilized antibodies to bind both the labeled and free lipoprotein to give a semi-quantitative result.
- U.S. Pat. No. 5,591,645 provides a chromatographic test strip with at least two portions. The first portion includes a movable tracer and the second portion includes an immobilized binder capable of binding to the analyte.
- Devices described herein generally include a strip of absorbent material (such as a microporous membrane), which, in some instances, can be made of different substances each joined to the other in zones, which may be abutted and/or overlapped.
- a strip of absorbent material such as a microporous membrane
- the absorbent strip can be fixed on a supporting non-interactive material (such as nonwoven polyester), for example, to provide increased rigidity to the strip.
- Zones within each strip may differentially contain the specific binding partner(s) and/or other reagents required for the detection and/or quantification of the particular analyte being tested for, for example, one or more molecules disclosed herein. Thus these zones can be viewed as functional sectors or functional regions within the test device.
- a fluid sample is introduced to the strip at the proximal end of the strip, for instance by dipping or spotting. The fluid migrates distally through all the functional regions of the strip. The final distribution of the fluid in the individual functional regions depends on the adsorptive capacity and the dimensions of the materials used.
- a lateral flow device is an analytical device having as its essence a test strip, through which flows a test sample fluid that is suspected of containing an analyte of interest.
- the test fluid and any suspended analyte can flow along the strip to a detection zone in which the analyte (if present) interacts with a capture agent (i.e. the polypeptide of the present invention) and a detection agent to indicate a presence, absence and/or quantity of the analyte.
- a capture agent i.e. the polypeptide of the present invention
- Numerous lateral flow analytical devices have been disclosed, and include those shown in U.S. Pat. Nos.
- lateral flow devices are one-step lateral flow assays in which a biological fluid is placed in a sample area on a bibulous strip (though non-bibulous materials can be used, and rendered bibulous, e.g., by applying a surfactant to the material), and allowed to migrate along the strip until the liquid comes into contact with a specific binding partner (such as an antibody) that interacts with an analyte (such as one or more molecules) in the liquid. Once the analyte interacts with the binding partner, a signal (such as a fluorescent or otherwise visible dye) indicates that the interaction has occurred.
- a specific binding partner such as an antibody
- analyte such as one or more molecules
- test strips can also incorporate control indicators, which provide a signal that the test has adequately been performed, even if a positive signal indicating the presence (or absence) of an analyte is not seen on the strip.
- control indicators which provide a signal that the test has adequately been performed, even if a positive signal indicating the presence (or absence) of an analyte is not seen on the strip.
- the construction and design of lateral flow devices is very well known in the art, as described, for example, in Millipore Corporation, A Short Guide Developing Immunochromatographic Test Strips, 2nd Edition, pp. 1-40, 1999, available by request at (800) 645-5476; and Schleicher & Schuell, Easy to Work with Bioscience, Products and Protocols 2003, pp. 73-98, 2003, 2003, available by request at Schleicher & Schuell Bioscience, Inc., 10 Optical Avenue, Keene, N.H. 03431, (603) 352- 3810; both of which are incorporated herein by reference.
- the method of the present invention is particularly suitable in diagnostic assays, in particular for the diagnosis of bacterial infections caused by the E coli 025b-ST131 clone. In some embodiments, the method of the present invention is particularly suitable for the diagnosis nosocomial infections and in particular, Hospital-acquired nosocomial infections.
- the method of the present invention is particularly suitable for the diagnosis of an infectious disease selected from the group consisting of cystic fibrosis, otitis media, keratitis, endophthalmitis, bacteremia, burn wound infection, pneumonia, meningitis, peritonitis or sepsis, more preferably pneumonia, meningitis, peritonitis or sepsis, and most preferably peritonitis or sepsis.
- the sample is a sample obtained from a patient who suffers from a bacterial infection.
- the patient is selected among immunocompromised and/or seriously ill patients in cancer centers, intensive care units, and organ transplant centres.
- the diagnostic method of the present invention is a valuable tool for practicing physicians to make quick treatment decisions.
- These treatment decisions can include the administration of an anti-bacterial agent (e.g. antibiotic) and decisions to monitor a subject for onset and/or advancement of the infection.
- the method disclosed herein can also be used to monitor the effectiveness of a therapy.
- the patient can be monitored while undergoing treatment using the methods described herein in order to assess the efficacy of the treatment protocol. In this manner, the length of time or the amount give to the patient can be modified based on the results obtained using the methods disclosed herein.
- 025b LPS extract inhibits bacteriophage LM33_P1 infection: appearance on agar plates.
- LPS extract from strain LM33 was mixed with bacteriophage LM33 P1 (left) or 536 P1 (right) at two different concentrations (10 5 and 10 4 pfu/mL) and assayed on two agar plates overlaid with an 025b strain (AVC02) or an 06 strain (536) as control. Enlargements of these two plates are shown to facilitate the observation.
- Bacterial strains used in this work belong to previously published collections of human commensal and extraintestinal E. coli gathered in France during the 2010s (13-15), from the ECOR collection (16) and the ColoColi collection (an ongoing French multicenter study collecting E. coli strains in the lower respiratory tract of mechanically ventilated patients).
- Phylogroup and ST belonging was determined as described in (17, 18).
- O-type and fimH allele were determined by PCR-based assays as previously described (19, 20), respectively. All strains were grown in lysogeny broth (LB) (DifcoTM Bacto-Tryptone 10 g/L, DifcoTM Yeast extract Difco 5 g/L, NaCl 5 g/L).
- Antibiotic susceptibility using the disk diffusion method was performed following the guidelines of the European Committee for Antimicrobial Susceptibility Testing guidelines.
- E. coli strains used for lipopolysaccharide (LPS) assays or bacteriophage susceptibility testing, are detailed below:
- LM33, LM36, AVC02 (ST131-025b:H4) and AVC03 (025b, non-ST131) are clinical strains responsible for ventilator-associated pneumonia
- Bacteriophages were isolated from sewage, using specific host.
- bacteriophages are named as follows: "host bacteria Px" (for example LM33 P1 represents the first bacteriophage isolated using strain LM33).
- bacteriophage solutions were purified using ultracentrifugation on cesium chloride gradient as previously described (22).
- spot test consisted in dropping off 10 of a growing liquid culture of the bacterial strain (OD 6 oonm 0.5) on an agar plate. After drying, 1 of the bacteriophage solution (LM33 P1, 10 7 pfu/mL) was added on one half of the bacterial drop. Plate was then incubated at 37°C during 4 hours before reading. A susceptible strain was identified by the presence of a crescent-shaped lysis area on the bacterial drop or the visualization of individualized plaques.
- EOP Efficiency of plaquing
- LPS extracts were purified from the same amount of bacteria (10 10 cfu) using a hot phenol-water-diethyl ether extraction (25) followed by extensive dialysis against sterile pyrolyzed water. High purity LPS was confirmed by performing agarose gel electrophoresis with ethidium bromide staining (nucleic acids detection) and SDS-PAGE 12% followed by Coomassie blue staining (proteins detection). Ten of each LPS extract were migrated on a SDS-PAGE 10% followed by silver staining to visualize the LPS O-antigen pattern (SilverSNAP Stain Kit II, Pierce).
- Adsorption assay and one-step growth were performed using LB (DifcoTM Bacto-
- Lysis kinetics were performed as detailed in the SI. Briefly, the growth of LM33 with and without LM33 P1 was followed overtime by recording optical density at 600 nm every 15 minutes.
- Aggregation assays were performed using 025 E. coli anti-serum (Statens Serum Institut, Copenhagen, Denmark) and observed under light microscope as detailed in the SI.
- LM33 P1 DNA was extracted from a purified bacteriophage solution, using DNase and R ase pretreatments followed by a phenol-chloroform extraction, modified from Pickard (27).
- LM33 genomic DNA was extracted using a Max Well Tissue DNA Purification kit (Promega, Madison, WI). Genomes annotation was performed by MicroScope plateform for strain LM33 and with RAST server for bacteriophage LM33 P1 (28, 29) followed by manual curation.
- Pneumonia was initiated by intranasal administration of lxlO 8 cfu of strain LM33 on anesthetized eight-week-old 25 g BALB/cJRj male mice (Janvier, Le Genest Saint Isle, France) as previously described (30). Mice were treated using bacteriophage LM33 P1 four hours post-infection, either by using the intranasal route (multiplicity of infection of 50, i.e. a ratio of viruses to bacteria equal to 50) or the intraperitoneal route (MOI of 500). Control mice received accordingly an intranasal or intraperitoneal identical volume of PBS (phosphate-buffered saline). Lungs were collected 17 hours post-infection on euthanized animals.
- PBS phosphate-buffered saline
- the septicemia model is essentially used to study intrinsic extraintestinal virulence of E. coli isolates (7).
- Four-week-old 17 g OF1 female mice (Janvier, Le Genest Saint Isle, France) were injected subcutaneously into the nape of the neck with lxlO 9 cfu of strain H1659 (ST131-025b:H4) (6).
- the single dose (MOI 60) was administered by intraperitoneal injection 2 hours post-infection while the double dose consisted in an injection (MOI 60) administered 2 and 12 hours post-infection.
- Control mice received an identical volume of PBS.
- Organs targeted by septic metastasis (heart- lung, spleen and liver) were collected on animals that died between 24 to 30 hours post-infection.
- the urinary tract infection model consists in a retrograde kidneys infection occurring after an intra-urethral injection of 5xl0 7 cfu of strain LM33 in the bladder, as previously described (31). Twenty-four hours after infection, 8-week-old 17 g CBA/j female mice (Charles River, Chatillon-sur-Chalaronne, France) were treated intraperitoneally with LM33 P1 (MOI of 200) while control mice received an identical volume of PBS. Kidneys were collected 48 hours post-infection.
- organs were mechanically homogenized in cold PBS using a gentleMACS Octo Dissociator (Milteny Biotec, Bergisch Gladbach, Germany) before being serially diluted and spread on Drigalski agar plates containing appropriate antibiotic to numerate colony. Bacteriophages count was performed on supernatant after centrifugation of homogenates according to routine methods.
- Bacteriophage LM33_P1 targets antibiotic resistant 025b E. coli strains.
- the E. coli strain LM33 isolated from an intensive care unit patient who developed a ventilator- associated pneumonia was used to isolate bacteriophage LM33 P1.
- Strain LM33 displays an 025b :H4 serotype, a B2 phylogroup (subgroup I) and a ST131 sequence-type as well as a multi-drug resistance phenotype with an extended spectrum beta-lactamase, a resistance to nalidixic acid, aminoglycosides (kanamycin, tobramycin, gentamicin, netilmicin excepted for amikacin where an intermediate phenotype is found), sulphonamides and chloramphenicol.
- the beta-lactam resistance is supported by a plasmid (pLM33) bearing the blaTEM-lc (penicillinase) and blaSHV-12 (extended spectrum beta-lactamase) genes, as well as by the bacterial chromosome containing the blaDHA-7 gene encoding a cephalosporinase and also a copy of the blaSHV-12 and blaTEM-lc gene (Table 1).
- pLM33 plasmid bearing the blaTEM-lc (penicillinase) and blaSHV-12 (extended spectrum beta-lactamase) genes, as well as by the bacterial chromosome containing the blaDHA-7 gene encoding a cephalosporinase and also a copy of the blaSHV-12 and blaTEM-lc gene (Table 1).
- Bacteriophage LM33_P1 is a Podoviridae distantly related to bacteriophage T7.
- Genome of bacteriophage LM33 P1 (38 979 bp; GC content of 50.8%; 49 ORFs predicted) lacks putative ORFs with homologies to integrase or recombinase.
- a BLAST analysis of the genomic sequence revealed that the four closest related bacteriophages were enterobacteria bacteriophages: three coliphages called PE3-1, KIF (33), EcoDSl (with 94% identity on > 88% of its length for all of them) and bacteriophage Dev2 infecting Cronobacter turicensis (with 83% identity on 85% of its length) (34). Alignment of these related bacteriophages with LM33 P1 revealed a similar spatial genome organization and confirmed the high homology between them (data not shown). Strikingly, the 5' extremity (the first 650 nucleotides) of the tail fiber gene is highly conserved in each bacteriophage genome, while the remaining part is highly divergent.
- the corresponding N- terminal region (IPR005604 / PF03906, InterPro / Pfam database) of this tail fiber protein is involved in its connection to the tail-tube (35) while the C-terminal part, involved in host recognition, often carries hydrolase activities as the endosialidase of bacteriophage KIF used for exopolysaccharide degradation (33, 36).
- BLAST searches on the C-terminal part of the tail fiber of bacteriophage LM33 P1 revealed homology to a domain belonging to the pectin lyase superfamily (IPRO 11050).
- Tridimensional structure prediction using Phyre 2 database (37) confirmed its close proximity to the endopolygalacturonase of Erwinia carotovora that belongs to the pectin lyase superfamily (100%) amino-acid predicted with a confidence >90%> for the tertiary structure, index of confidence for homologous protein 94.1%, Protein Data Bank entry: 1BHE).
- Bacteriophage LM33_P1 is highly efficient and rapid in vitro. Adsorption of LM33 P1 bacteriophage on its host is fast with > 90% of the viral population attached to cells after 3.5 minutes with an adsorption constant of 1.2x10 ⁇ 8 mL/min. Newly produced virions are detected within the bacteria as soon as 7 minutes post-infection (eclipse period) while host lysis occurs in 9 minutes (latent period) with a burst size of 317 (95 % confidence interval: 289-345) (data not shown).
- Bacteriophage LM33_P1 specifically binds to 025b LPS O-antigen. Host range of bacteriophage LM33 P1 strongly suggested that O-chain of LPS could be involved in its specificity. Using LPS competition assays we observed that purified LPS from strain LM33 was able to partially inhibit interaction between bacteriophage LM33 P1 and strain LM33 as well as other 025b strains (Table 2).
- LPS extract from 025b strain was also reducing infectivity of bacteriophage LM33 P1 on liquid medium in a dose dependent manner (data not shown), while LPS extracts from 06 and 025a strains had no effect.
- Adsorption of bacteriophage LM33_P1 is hindered by capsule production.
- Bacteriophage LM33_P1 efficiently infects its host in vivo. As bacteriophage LM33_P1 exhibited impressive in vitro characteristics, we investigated its in vivo activity in three different animal infection models relevant to ST131 clinical epidemiology: pneumonia, septicemia and urinary tract infection ( Figures 2-4). Since strain LM33 was isolated from a patient with pneumonia, we first attempted to establish pneumonia in mice. Using an inoculum 50 times higher than previously reported in such model (30) and despite clear macroscopic lung lesions, strain LM33 was not lethal preventing us to use survival as an indicator of bacteriophage efficacy. We therefore evaluated LM33 P1 efficacy by counting bacteria from lung homogenates collected 17 hours following infection.
- mice were treated 4 hours post-infection either by control solution (PBS), intranasal (MOI 50) or intraperitoneal (MOI 500) bacteriophages.
- PBS control solution
- MOI 50 intranasal
- MOI 500 intraperitoneal
- bacteriophage LM33 P1 Intraperitoneal administrations of bacteriophage LM33 P1 (MOI 60, single dose at H2 post- infection or two doses at H2 and HI 2 post-infection) were not sufficient to prevent animals death.
- bacteria and bacteriophages content was analyzed: i) in liver, spleen and lung- heart homogenates of bacteriophage-treated groups the number of bacteria was reduced compared to control group; ii) two doses appeared to be more efficient than a single one, reaching a significant reduction of approximately 1.4 Logio (median bacterial count decrease from 8.5xl0 6 to 2.9xl0 5 in heart-lungs, 7.7xl0 5 to 3.2xl0 4 in the liver and 3.5xl0 5 to 1.4xl0 4 cfu/g in the spleen); iii) bacteriophage counts were in the same order of magnitude in all organs, but were significantly higher when two
- mice received a single bacteriophage treatment intraperitoneally (MOI of 200).
- MOI of 200
- a 2 Logio reduction of the bacterial load was observed in the kidneys in the treated group compared to control (1.5xl0 5 vs 8.8xl0 2 cfu/g, p ⁇ 0.001).
- Antibiotic resistance is a public health problem worldwide. In less than 10 years, multi-drug resistant ST131-025b:H4 E. coli clonal complex have spread over the planet, now being present in both animals and humans (2). Unfortunately, the discovery of new antibiotics did not turn out to be as successful as initially expected, leading to the reappraisal of phage therapy.
- One of the main advantages of bacteriophages often reported is their specificity to infect few strains within a species, having then a limited impact on patient's microbiota. Along with monoclonal antibodies (anti-025b antibodies have been proven to exert a protective effect in mouse septicemia model) (39), bacteriophages are the only anti-infectious tools that could reach such specificity.
- Bacteriophage LM33 P1 specificity was linked to the 025b O-antigen and not to the sequence type (i.e. none of the non-025b ST131 strains were susceptible to bacteriophage LM33 P1 while all 025b-ST69 strains tested were susceptible). Furthermore, susceptibility of ST131-025b:H4 strains to bacteriophage LM33 P1 was independent of the fimH allele, a marker of the epidemiologic evolution of this clone (32).
- bacteriophage LM33 P1 was unable to infect 025a strains, despite a highly similar O-antigen structure where polysaccharides repeated units only differ by one monosaccharide (fucose versus rhamnose), a fine discrimination that is not possible with classical antibodies used for serotyping until the recent description of 025b monoclonal antibodies (21).
- bacteriophage LM33 P1 can mask phage receptors by the production of extracellular exopolysaccharides (capsules), which can also help bacteria escaping immune cells recognition (52, 53).
- capsules extracellular exopolysaccharides
- bacteriophage LM33 P1 coverage increased to 80% among all ST131-025b:H4 strains and to 73% among all 025b strains tested.
- bacteriophages pharmacokinetic is highly complex, due to their intrinsic properties (bacteria-driven self-expansion, diffusion, adsorption, threshold to prime a viral expansion, etc.) (61-63) and cannot be compared to traditional pharmacokinetic of antibiotics.
- the curative dose applied is always related to the initial known dose of pathogenic bacteria, which is therefore a gross estimation of what is needed (amount of bacteria could be highly different between time of inoculation and treatment due to bacterial growth). Consequently, our data should not be over-translated to the clinical setting.
- bacteriophages including LM33 P1 as shown in this study, can quickly reduce the load of their host within a complex environment including the gut of mammals (64).
- Our data also support higher efficacy when bacteriophages are applied locally (intranasal instillation to treat pneumonia) than when used via a systemic administration.
- such bacteriophages could be used as a selective antimicrobial agent for controlling passive carriage of ST131-025b:H4 strains in human gut in order to reduce its dissemination, particularly in healthcare-associated environments.
- E. coli strains residing in the digestive tract constitute a well-known reservoir for urinary tract infections but probably also for ventilator-associated pneumonia (14).
- bacteriophage LM33 P1 or proteins from it offer opportunities to develop several tools.
- the tail fiber could be used to kill specifically 025b E. coli strains using bacteriocins, as previously shown for 0104 E. coli strains involved in enterohemorragic colitis (66).
- Other approaches could be foreseen where bacteriophages are reprogrammed and could suppress antibiotic resistance genes using CRISPR-Cas system (67) or express well-chosen beneficial enzymes to fight biofilm (68). Deeper investigations on the infectious cycle of this bacteriophage are now required to determine which molecular mechanisms are responsible for its fast-killing component.
- Bacteriophage LM33 P1 could also be used from now as a starting platform to develop highly virulent synthetic bacteriophages with various host specificity (69).
- Table 1 Main genotypic characteristics of strain LM33 and plasmid pLM33.
- Genome size GC content: 51.5 % Number of genes: 5 450 287 bp 5276
- iroN Enterobactin siderophore receptor protein
- fyuA siderophore
- prfB P -related fimbriae regulatory gene
- papC P fimbriae
- traT serum resistance-associated outer membrane papGIII (P fimbriae) protein
- gad glutamate decarboxylase
- mchF ABS transporter protein
- Beta-lactam resistance blaDHA-7, blaSHV-12, blaTEM-lC
- Plasmid pLM33 accession number: PRJEB9970
- Beta-lactam resistance blaSHV-12, blaTEM-lC
- Table 2 Data obtained during plaque test inhibition assays with different LPS extracts and randomly chosen couples of viruses-bacteria. (+)/(-): presence/absence of an inhibitory effect of LPS extract, - : not tested.
- LF82_P2 C RY09 (04) (-) - - a described in (30), b bacteriophages isolated using ventilator-associated pneumonia (VAP) strains (423, 416) and active on others VAP strains (HI 7, LM49), 0 bacteriophage isolated using an adherent-invasive E. coli (LF82) and active on VAP strain RY09.
- VAP ventilator-associated pneumonia
- LF82 0 bacteriophage isolated using an adherent-invasive E. coli
Abstract
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US6232107B1 (en) | 1998-03-27 | 2001-05-15 | Bruce J. Bryan | Luciferases, fluorescent proteins, nucleic acids encoding the luciferases and fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items |
US6699722B2 (en) | 2000-04-14 | 2004-03-02 | A-Fem Medical Corporation | Positive detection lateral-flow apparatus and method for small and large analytes |
US20040241876A1 (en) | 2000-12-22 | 2004-12-02 | France Fannes | Flow through assay device, diagnostic kit comprising said assay device and use of said assay device in the detection of an analyte present in a sample |
US7531362B2 (en) | 2001-06-07 | 2009-05-12 | Medmira Inc. | Rapid diagnostic assay |
WO2008030546A2 (en) | 2006-09-06 | 2008-03-13 | Ouantrx Biomedical Corporation | Lateral flow test strip with migrating label |
WO2013045863A1 (en) * | 2011-09-29 | 2013-04-04 | Institut Pasteur | Detection of e.coli strains of serotype 0104 |
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2017
- 2017-04-12 US US16/093,202 patent/US20190120838A1/en not_active Abandoned
- 2017-04-12 ES ES17718047T patent/ES2866880T3/en active Active
- 2017-04-12 EP EP17718047.8A patent/EP3442993B1/en active Active
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EP3442993B1 (en) | 2021-01-20 |
WO2017178554A1 (en) | 2017-10-19 |
ES2866880T3 (en) | 2021-10-20 |
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